Paola Cuneo

The control of cellular motility and the role of gelsolin

Solation of actin gel by gelsolin is much less efficient in the presence of a high concentration of macromolecular solutes. The rigidity of the gel formed by 12 μM actin is lowered from 4 to 0.33 dynes/cm2 by 15 nM gelsolin, while in 6% (w/v) polyethylene glycol, rigidity is lowered only from 20 to 11 dynes/cm2 by 64 nM gelsolin. Owing to the large concentration of protein, transitions in the fluid‐ and gel‐like properties of the cytoplasm are expected to be problematic when promoted by gelsolin alone.

The actin gelling activity of chicken gizzard α-actinin at physiological temperature is triggered by water sequestration

At 37°C, in the presence of 6% ( ) polyethylene glycol 6000, 30 nM α‐actinin from chicken gizzard induces the gelation of 12 μM actin. Static measurement shows that the addition of 30 nM α‐actinin increases the rigidity of the system from 23.5 to 54 . According to the theory of osmoelastic coupling, also large additives, such as the proteins of the cell sap, are able to cause an osmotic stress equivalent to that caused by polyethylene glycol. We thus conclude that, in vivo, α‐actinin acts as an actin gelling protein.

Protein cross talking through osmotic work: the free energy of formation of the MgADP-myosin complexes at the muscle protein osmotic pressure

A method is presented to determine the energy of formation of the myosin-ADP complexes at the muscle protein osmotic pressure. It is found that, at 18 kP, the putative protein osmotic pressure in skeletal muscle, the increase of MgADP from 0.05 to 2 mmolal, increases the free energy of myosin-ADP and of myosin-(ADP)2 by 0. 756 and by 9.85 kJ/mol, respectively, and decreases the free energy of myosin by 8.34 kJ erg/mol. It is pointed out that the local changes of water chemical potential, induced by the binding of MgADP to myosin, can be sensed by other structures of the contractile machinery, which per se may even be insensitive to MgADP. Cross talking between macromolecules can thus be achieved by changes of the water chemical potential.

Preferential binding of α-actinin to actin bundles

At 37°C, the α‐actinin‐F‐actin binding isotherm is anomalous. In 6.7% polyethylene glycol 6000, concomitantly with the formation of actin bundles, the binding isotherm becomes hyperbolic (K diss. = 11.3 μM). α‐Actinin increases the rigidity of the networks formed by actin bundles in polyethylene glycol and by paracrystalline actin in 16 mM MgCl2 but not by F‐actin. It is proposed that in the cell α‐actinin functions are mostly carried on by interaction with actin bundles.

Dissecting the free energy of formation of the 1:1 actomyosin complex

The behaviour of solutions of pure myosin, of pure F-actin and of the equimolar mixture of myosin and of F-actin is studied. It is found that the chemical potential of the two proteins, in separate solutions, increases monotonically with the increase of protein osmotic pressure. A method is presented to determine the chemical potential of the 1:1 actin-myosin complex formed from equimolar solutions of myosin and of F-actin (as monomer). This is the first evaluation of the chemical potential of actomyosin under conditions similar to those of skeletal muscle. It is found that the filament suspensions of myosin and of the 1:1 actin-myosin complex display a high non-ideal behavior as well as distinctly different energy profiles as a function of protein osmotic pressure. This supports the hypothesis that, in muscle: (a) detached cross-bridge change significantly their free energy when sarcomere is shifting from the relaxed to the active or to the rigor state; and (b) the cross-bridge attachment-detachment process is accompanied by changes of muscle protein osmotic pressure.

Is nebulin truly a component of the thin filament

Thin filaments were prepared from rabbit and beef skeletal muscle with three different procedures, both at high and low ionic strength. Nebulin was always found to be associated with the myosin fraction and was always absent from the thin filament fraction.